A first step in sample analysis typically involves collecting the sample. For example, a first step in a biological analysis such as RNA gene expression profiling or protein biomarker profiling is to collect a particular sample so that its biochemical constituents can be analyzed. However, prior to analysis, a solid sample specimen, typically, is prepared by deconstructing it into a plurality of smaller fragments of the specimen to enable more accurate analysis.
Recently, downstream analytical processing of samples has undergone significant improvements, including with regard to sensitivity, throughput and like. Due to these significant improvements in downstream processes, deficiencies in upstream sample collection and preparation have become more apparent. One upstream processing enhancement has been the ultrasonic systems and methods for treating a sample described in co-pending, co-owned U.S. patent application Ser. No. 10/777,014, entitled Apparatus and Methods for Controlling Sonic Treatment,” the entire disclosure of which is incorporated by reference.
A challenge of sample preparation is that the types of samples are diverse. For example, samples may be biological, non-biological or a combination thereof. They may be from animals or plants. Samples may include, without limitation, cells, tissues, organelles, bones, seeds, chemical compounds, minerals, metals, or any other material for which analysis is desired.
Sample preparation is particularly challenging for solid biological samples, such as tissue samples. Physical and/or chemical approaches are often employed to disrupt and homogenize the solid sample for biochemical extraction. While appearing deceptively simple, transitioning a sample of biologically active tissue, for example, on the order of 1 gram, to a plurality of biomolecules that are stabilized and isolated in an appropriate analytical solution is exceedingly complex, very difficult to control, and prone to introduction of errors and/or sample constituent degradation.
Another challenge associated with sample preparation relates to the lability of the target molecules. For some applications, an overriding criterion is to retain the native biochemical environment prior to sample collection and throughout the extraction process, without perturbing the biochemical constituents to be analyzed. For example, RNases are extremely robust and may significantly degrade the mRNA profile of a tissue sample if the RNases are not immediately stabilized (typically thermal or chemical inactivation) at the time of tissue collection and during sample processing or homogenization. Often, to minimize perturbation of the biochemical profile of the sample, the tissue is flash-frozen (e.g., via direct immersion of the sample following procurement in liquid nitrogen) and stored at cryogenic temperatures (e.g., −80° C. or lower), which inhibits degradative processes.
Conventionally, once a sample is stabilized from thermal and/or chemical degradation, it is pulverized in liquid nitrogen at a temperature of about −196° C., for example, using a mortar and pestle. Other pulverizing systems available include a rotor-stator (polytron) and a bead-beater apparatus, which do not operate at cryogenic temperatures. In a typical example, a frozen specimen having a volume of approximately 1 cm3 may be fragmented into a plurality of solid fragments each having a volume of approximately 100 um3 or less.
Prior art approaches for performing such fragmentation suffer from many drawbacks. One such drawback is that liquid nitrogen is difficult and hazardous to work with. Another drawback is that prior art approaches can be slow and tedious. A further drawback is they involve direct contact between the sample and the fragmenting agents. For example, the sample, typically, is not contained during fragmentation causing portions of the sample to be deposited on the fragmenting devices in a non-recoverable manner. This, in turn, causes reduced sample recovery and extensive apparatus cleaning between operating cycles. Also, such deposits cause increased operator exposure to potentially hazardous samples, and/or the sample may be degraded by enzymes, bacteria, fungi, or other external contaminants.
Another challenge to sample preparation is maintaining the sample at an appropriate temperature. A disadvantage of the direct contact prior art devices, particularly the automated prior art devices, is that the sample may become sufficiently heated to cause the sample to degrade. This disadvantage is accentuated by sample fragmentation due to increased thermo-sensitivity resulting from increased surface area. Thawing also makes it difficult to transfer sample particles to a vessel for further processing. Another drawback is that conventional techniques have size range limitations. For example, a high percentage of a 25-mg sample would be lost in a 5-ml bead-beating system, causing unacceptably low sample recovery.
Accordingly, there is a need for an improved approach to preparing samples for further analysis.